Application of developed building blocks in the construction of genetic manipulation vectors.

<p>(A) Diagram showing the TGRV and EEV constructed for the adenylate kinase 4 (TcADK4) gene. I, pGEM-T Easy with a cloned genomic fragment comprising <i>TcADK4</i>, ISs (solid bold lines) and flanking coding sequences (light grey boxes). II, TGRVs produced by replacement of a fragment of <i>TcADK4</i> with the SMs of p<i>Tc</i>R-HG Hyg - and p<i>Tc</i>R-GA Neo -. For clarity equivalent sequences in both constructs are only shown for pL4-GA Neo. III, The TGRVs were linearized and independently transfected into epimastigote cells (arrow). The homologous recombination events between the constructs and the genomic locus are represented with crossed dotted lines. IV, resulting recombinant loci and expected amplification products. Primers 4cF and 4cR bind at positions 382 and 440 in the upstream and downstream genes respectively. Similarly HR, gF, gR and aF are primers specific for the HX1, gapdh II, gapdh I-II and actin ISs. The systematic ID of the flanking genes is given below. V, EEV generated by insertion of the SM of p<i>Tc</i>R-HG Neo - upstream the cloned fragment containing <i>TcADK4</i>. VI, Tagged EEV obtained after in-frame insertion of eGFP in replacement of a fragment of <i>TcADK4</i>. The numbers are the lengths, in base pairs, of the respective element above, the amplification products (in I and IV) or targeting sequences (in III). The letters represent the restriction enzyme sites used in the construction steps. N, NsiI; S, SalI; B, BamHI; C, Csp45I. (B) PCR analysis of genomic DNA from transgenic (T) and wild type (W) epimastigotes. The constructs and primer pairs employed in each case are indicated as well as the relevant lengths of the molecular weight marker bands. (C) Western blot analysis of extracts from wild type (W) and transgenic parasites obtained with pL4x-HG Neo (L4), pL4x-GFP HG Neo (L4G) and pTEXL-Neo (G). Transgenic parasites were cultured with 100 µg/mL of G418. Top panels derive from membranes stained with anti-<i>Tc</i>ADK4 (left) or anti-GFP (right) while bottom panels are equivalents stained with anti-PAR2. Below the lanes, <i>Tc</i>ADK4 or GFP expression levels relative to those of PAR2 are indicated. Molecular weights are shown.